Devin T. Edwards

Hidden dynamics in the unfolding of individual bacteriorhodopsin proteins

Pulling apart protein unfolding Elucidating the details of how complex proteins fold is a longstanding challenge. Key insights into the unfolding pathways of diverse proteins have come from single-molecule force spectroscopy (SMFS) experiments in which proteins are literally pulled apart. Yu et al. developed a SMFS technique that could unfold individual bacteriorhodopsin molecules in a native lipid bilayer with 1-µs temporal resolution (see the Perspective by Müller and Gaub). The technique delivered a 100-fold improvement over earlier studies of bacteriorhodopsin and revealed many intermediates not seen before. The authors also observed unfolding and refolding transitions between intermediate states. Science, this issue p. 945; see also p. 907 Mechanical unfolding of a membrane protein reveals previously undetected intermediates and equilibrium refolding. Protein folding occurs as a set of transitions between structural states within an energy landscape. An oversimplified view of the folding process emerges when transiently populated states are undetected because of limited instrumental resolution. Using force spectroscopy optimized for 1-microsecond resolution, we reexamined the unfolding of individual bacteriorhodopsin molecules in native lipid bilayers. The experimental data reveal the unfolding pathway in unprecedented detail. Numerous newly detected intermediates—many separated by as few as two or three amino acids—exhibited complex dynamics, including frequent refolding and state occupancies of <10 μs. Equilibrium measurements between such states enabled the folding free-energy landscape to be deduced. These results sharpen the picture of the mechanical unfolding of membrane proteins and, more broadly, enable experimental access to previously obscured protein dynamics.

Optimizing 1-μs-Resolution Single-Molecule Force Spectroscopy on a Commercial Atomic Force Microscope

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is widely used to mechanically measure the folding and unfolding of proteins. However, the temporal resolution of a standard commercial cantilever is 50–1000 μs, masking rapid transitions and short-lived intermediates. Recently, SMFS with 0.7-μs temporal resolution was achieved using an ultrashort (L = 9 μm) cantilever on a custom-built, high-speed AFM. By micromachining such cantilevers with a focused ion beam, we optimized them for SMFS rather than tapping-mode imaging. To enhance usability and throughput, we detected the modified cantilevers on a commercial AFM retrofitted with a detection laser system featuring a 3-μm circular spot size. Moreover, individual cantilevers were reused over multiple days. The improved capabilities of the modified cantilevers for SMFS were showcased by unfolding a polyprotein, a popular biophysical assay. Specifically, these cantilevers maintained a 1-μs response time while eliminating cantilever ringing (Q ≅ 0.5). We therefore expect such cantilevers, along with the instrumentational improvements to detect them on a commercial AFM, to accelerate high-precision AFM-based SMFS studies.

Rapid Characterization of a Mechanically Labile α-Helical Protein Enabled by Efficient Site-Specific Bioconjugation

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize mechanical properties of biomolecules. Historically, accessibility relies upon the nonspecific adhesion of biomolecules to a surface and a cantilever and, for proteins, the integration of the target protein into a polyprotein. However, this assay results in a low yield of high-quality data, defined as the complete unfolding of the polyprotein. Additionally, nonspecific surface adhesion hinders studies of α-helical proteins, which unfold at low forces and low extensions. Here, we overcame these limitations by merging two developments: (i) a polyprotein with versatile, genetically encoded short peptide tags functionalized via a mechanically robust Hydrazino-Pictet-Spengler ligation and (ii) the efficient site-specific conjugation of biomolecules to PEG-coated surfaces. Heterobifunctional anchoring of this polyprotein construct and DNA via copper-free click chemistry to PEG-coated substrates and a strong but reversible streptavidin-biotin linkage to PEG-coated AFM tips enhanced data quality and throughput. For example, we achieved a 75-fold increase in the yield of high-quality data and repeatedly probed the same individual polyprotein to deduce its dynamic force spectrum in just 2 h. The broader utility of this polyprotein was demonstrated by measuring three diverse target proteins: an α-helical protein (calmodulin), a protein with internal cysteines (rubredoxin), and a computationally designed three-helix bundle (α3D). Indeed, at low loading rates, α3D represents the most mechanically labile protein yet characterized by AFM. Such efficient SMFS studies on a commercial AFM enable the rapid characterization of macromolecular folding over a broader range of proteins and a wider array of experimental conditions (pH, temperature, denaturants). Further, by integrating these enhancements with optical traps, we demonstrate how efficient bioconjugation to otherwise nonstick surfaces can benefit diverse single-molecule studies.

Optimizing force spectroscopy by modifying commercial cantilevers: Improved stability, precision, and temporal resolution

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) enables a wide array of studies, from measuring the strength of a ligand-receptor bond to elucidating the complex folding pathway of individual membrane proteins. Such SMFS studies and, more generally, the diverse applications of AFM across biophysics and nanotechnology are improved by enhancing data quality via improved force stability, force precision, and temporal resolution. For an advanced, small-format commercial AFM, we illustrate how these three metrics are limited by the cantilever itself rather than the larger microscope structure, and then describe three increasingly sophisticated cantilever modifications that yield enhanced data quality. First, sub-pN force precision and stability over a broad bandwidth (Δf=0.01-20Hz) is routinely achieved by removing a long (L=100μm) cantilevers gold coating. Next, this sub-pN bandwidth is extended by a factor of ∼50 to span five decades of bandwidth (Δf=0.01-1000Hz) by using a focused ion beam (FIB) to modify a shorter (L=40μm) cantilever. Finally, FIB-modifying an ultrashort (L=9μm) cantilever improves its force stability and precision while maintaining 1-μs temporal resolution. These modified ultrashort cantilevers have a reduced quality factor (Q≈0.5) and therefore do not apply a substantial (30-90pN), high-frequency force modulation to the molecule, a phenomenon that is unaccounted for in traditional SMFS analysis. Currently, there is no perfect cantilever for all applications. Optimizing AFM-based SMFS requires understanding the tradeoffs inherent to using a specific cantilever and choosing the one best suited to a particular application.

Improved Force Spectroscopy Using Focused-Ion-Beam-Modified Cantilevers

Atomic force microscopy (AFM) is widely used in biophysics, including force-spectroscopy studies of protein folding and protein-ligand interactions. The precision of such studies increases with improvements in the underlying quality of the data. Currently, data quality is limited by the mechanical properties of the cantilever when using a modern commercial AFM. The key tradeoff is force stability vs short-term force precision and temporal resolution. Here, we present a method that avoids this compromise: efficient focused-ion-beam (FIB) modification of commercially available cantilevers. Force precision is improved by reducing the cantilevers hydrodynamic drag, and force stability is improved by reducing the cantilever stiffness and by retaining a cantilevers gold coating only at its free end. When applied to a commonly used short cantilever (L=40μm), we achieved sub-pN force precision over 5 decades of bandwidth (0.01-1000Hz) without significantly sacrificing temporal resolution (~75μs). Extending FIB modification to an ultrashort cantilever (L=9μm) also improved force precision and stability, while maintaining 1-μs-scale temporal resolution. Moreover, modifying ultrashort cantilevers also eliminated their inherent underdamped high-frequency motion and thereby avoided applying a rapidly oscillating force across the stretched molecule. Importantly, fabrication of FIB-modified cantilevers is accessible after an initial investment in training. Indeed, undergraduate researchers routinely modify 2-4 cantilevers per hour with the protocol detailed here. Furthermore, this protocol offers the individual user the ability to optimize a cantilever for a particular application. Hence, we expect FIB-modified cantilevers to improve AFM-based studies over broad areas of biophysical research.

High Precision AFM-Based SMFS of Mechanically Labile T3SS Effectors

an aqueous medium without the need for external potentials. We also demonstrate of a single gold nanoparticle trapped by a scanning aperture can be used as a nano-antenna for plasmonic enhancement of fluorescence in fluidic environment. Our trapping approach with appropriately tailored geometry might allow us to trap and levitate single proteins or macromolecules even in high salt concentrations which could open doors to well-controlled studies at the single-molecule level.

Custom Modification of AFM Tips for Fast, High Force Resolution Single-Molecule Force Spectroscopy

In addition to providing the ability to image on the nanoscale, atomic force microscopy (AFM) has the ability to measure small (pN) forces. This ability has led to new insights into conformational changes in biological molecules; in particular, single-molecule force spectroscopy (SMFS) is a powerful tool to investigate folding in proteins. Ideally, one could observe folding in proteins at time scales in the microsecond range with both short-term force precision and long-term force stability. Recent work [1] has shown that to minimize force drift, one must use a soft AFM cantilever due to instrumental noise. Such soft, long cantilevers have poor temporal resolution. When high temporal resolution is required, one choses a shorter cantilever, which has a higher spring constant (k ∝ L) and hence increased force drift. Recently, it has been shown that by modifying a commercially available cantilever with focused ion beam (FIB) milling, short AFM cantilevers (L~40 μm) can be softened to reduce force drift, without sacrificing temporal resolution. The resulting cantilevers offer state-of-the-art force stability and precision, with temporal resolution ~70 μs [2]. To further improve the performance of modified AFM cantilevers, FIB modification strategies need to be developed to extend the technique to ultra-small cantilevers (L~9 μm) , which offer temporal resolution ~1 μs [3].We present several modification strategies, each of which decrease the stiffness of the resultant cantilevers while retaining fast response times. Additionally, we explore techniques that improve the yield of the process.